Phosphatidylinositol (3,5)-bisphosphate (PI3,5P2), a relatively unstudied signaling lipid, is required in all eukaryotes and cell-types tested. Defects in PI3,5P2 levels specifically lead to profound neurological defects. For example, a significant fraction of amylotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS) patients have heterozygous mutations in Fig4, a protein that functions in the synthesis and turnover of PI3,5P2. Moreover, selected mutations in Fig4 lead to a form of Charcot-Marie Tooth syndrome that is early onset and causes paraplegia/quadriplegia. Consistent with these human diseases, we found that depletion of PI3,5P2 leads to neurological defects and embryonic lethality in mice. These studies and others predict that defects in PI3,5P2 metabolism underlie many neurodegenerative disorders. In order to identify and ultimately treat these diseases, we must first determine how the levels of this lipid are regulated, as well as determine the pathways that are regulated by PI3,5P2. While the long-term goal is to treat human disease, the yeast, Saccharomyces cerevisiase, provides unique advantages. Most of the machinery that regulates PI3,5P2 levels is conserved from yeast to humans. In addition, our newly identified candidate pathways directly upstream and downstream of PI3,5P2, are conserved as well. Thus, results from yeast studies will likely provide significant insights into PI3,5P2 function and regulation in humans. Importantly, the versatility of classical and global approaches to yeast genetics combined with the facility of performing microscopy and biochemistry on large numbers of cells, will enable us to rapidly identify and assign functions to proteins required for these pathways. Our goals are to: 1) Determine how the Vac14 complex functions in both the synthesis and turnover of PI3,5P2. 2) Determine how the cyclin, Pho80 and its kinase Pho85, act upstream of the Vac14 complex to regulate PI3,5P2 levels. 3) Complete a global screen designed to identify downstream molecular targets and pathways that are regulated by PI3,5P2, and then choose selected targets including the unexpected target, TORC1, to determine the how regulation by PI3,5P2 is achieved.

Public Health Relevance

Phosphatidylinositol (3,5)-bisphosphate (PI3,5P2), is required in all cell-types tested. However defects in PI3,5P2 levels most acutely affect neural cells, and low levels of PI3,5P2 lead to severe neurological disorders. The overall goals of this application are to 1) Determine the mechanisms the regulate PI3,5P2 levels, 2) Identify upstream pathways that signal the stimulus-induced acute changes in PI3,5P2, 3) Identify and characterize the downstream pathways that require PI3,5P2 for their function. Results of these studies should provide new insights into the roles and regulation of PI3,5P2 and will provide a basis for determining to roles of PI3,5P2 in the nervous system.